The objective of this research was to identify SNPs and to efficiently develop an Infinium iSelect assay which contained over 50,000 SNPs from soybean.  A total of 498,921,777 reads of 35-45bp in length were obtained from sequencing reduced representation libraries created from several soybean accessions which included the wild soybean genotypes PI468916 and PI479752 and the cultivated soybean lines, Essex, Evans, Archer, Minsoy, Noir 1, and Peking.  These reads were mapped to the soybean whole genome sequence and identified 188,272 SNPs.  After applying several filters, a total of 146,161 of the 188,272 SNPs were ideal candidates for Infinium design.  In order to maximize the distance between selected SNPs along each chromosome, increase assay success rate, and minimize the number of SNPs with low minor allele frequency, an iterative selection algorithm based on a selection index was developed which was used to select 60,800 SNPs for Infinium assay design.  Of the 60,800 SNPs, 50,701 were targeted to euchromatic regions and 10,000 to heterochromatic regions of the 20 soybean chromosomes.  In addition, 99 SNPs were targeted to unanchored sequence scaffolds.  Of the 60,800 SNPs a total of 52,041 passed Illumina’s manufacturing phase to produce the SoySNP50K iSelect SNP chip.  Validation of the SoySNP50K chip with 96 landrace genotypes and 96 elite cultivars showed that 45,735 SNPs were polymorphic.  In addition 74% and 65% of the SNPs had minor allele frequencies >10% among the landraces and elite cultivars, respectively.  The SoySNP50K iSelect SNP chip was used to genotype over 1000 RILs from both Williams 82 x Essex and Williams 82 x PI479752 populations. The resulting high resolution linkage maps should improve the soybean whole genome sequence assembly (Glyma1.01).